1. Field of the Invention
The present invention is directed to chemical biosensors or more particularly to fixed parallel plate chemical biosensors and capacitor arrays and methods of making same utilizing immobilized monoclonal half-antibodies.
2. Description of the Prior Art
In normal physiological development, VEGF is a crucial regulator of vascular development during embryogenesis (vasculogenesis) and blood-vessel formation in the adult (angiogenesis). The members of the VEGF and VEGF-receptor protein families have distinct but overlapping ligand-receptor specificities, cell-type expression, and function. VEGF-receptor activation in turn regulates a network of signaling processes in the body that promote endothelial cell growth, migration, and survival. VEGF also plays a critical role in the pathological angiogenesis that occurs in a number of diseases, including cancer. VEGF and the Flk-1/KDR RTK have been implicated as the key endothelial cell-specific factor signaling pathway required for pathological angiogenesis, including tumor neovascularization. In tumor progression, activation of VEGF pathways promotes tumor vascularization, facilitating tumor growth and metastasis. Abnormal VEGF function is also associated with other diseases including atherosclerosis, psoriasis, age related macular degeneration, diabetic blindness, rheumatoid arthritis, and hyperthyroidism.
Advances in understanding the biology of angiogenesis have led to the development of several therapeutic modalities for the inhibition of the VEGF tyrosine kinase signaling pathway. Inhibition of the VEGF tyrosine kinase signaling pathway blocks new blood vessel formation in growing tumors, leading to stasis or regression of tumor growth. The growth of human tumors and development of metastases depend on the de novo formation of blood vessels to reach and provide nutrients for the hypoxic tumor microenvironment. The formation of new blood vessels is tightly regulated by specific growth factors that target receptor tyrosine kinases (RTKs). Initial attempts to block VEGF by using the humanized monoclonal antibody bevacizumab (Avastin®, Genentech/Roche), and two kinase inhibitors sorafenib (Nexavar; Bayer) and sunitinib (Sutent, Pfizer) targeting the VEGF receptor (VEGF-R) tyrosine kinases are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade for neurological cancers. A number of these modalities are under investigation in clinical studies to evaluate their potential to treat various forms of human cancer, but the ability of such studies are limited by the fact that local, real-time in vivo measurement of the VEGF levels and the trends of the VEGF transduction are not readily available.
Biosensors which continuously monitor their surroundings to provide background statistics and warnings against unhealthy conditions are known to be useful in medical technology. In the proposed applications, micro-scale solutions are sought for to minimize cost, minimize impact, and maximize useful lifetime of the biosensor. One-time use biosensors are of limited utility due to the inherent need to monitor the process of the tumor growth and elimination for the duration of treatment.
The prior art discussion on the subject matter of “Biosensors” is extensive and far reaching. There are numerous examples of biosensors (e.g. gravimetric biosensors). The basis of detection is the decrease in the resonant frequency of a resonator that occurs as analyte species attached to the resonating element. Analyte specificity is conferred for biological analytes by functionalizing (treating) the exposed surface of the resonator with ligands that recognize and bind to the target analyte species. Examples of suitable binding entities for target biological analytes include antibodies, receptors, lectins, aptamers and oligonucleotides.
One type of biosensor presented by prior art is the gravimetric biosensor, wherein the immobilized binding group is located in one or more areas on the surface of a membrane. The immobilized binding group locations', size, area, and immobilization density are designed to maximize the observed frequency and/or amplitude shifts on the membrane by the target analyte binding. This, in turn, maximizes the discrimination observable by subsequent frequency and/or amplitude shifts on the membrane based upon all combinations of specific and non-specific binding. This discrimination may take three forms: (a) change in resonant frequency of the membrane, (b) appearance or disappearance of higher order harmonic vibrations, or (c) change in amplitude decay rates. In such a biosensor, a single membrane may be comprised of a plurality of individually addressable elements for actuation and for sensing purposes. That permits the specific excitement of selected higher order vibrational modes and enables simultaneous vibration actuation of an alarm circuit or like devices. The principles of acoustic wave analysis, which can be utilized in a gravimetric sensor, is well known and has appeared in literature for more than a decade.
Molecular interactions can be detected electronically through the polarizability of biological macromolecules, optically through the use of fluorescencing tags, radiometrically through the use of radioactive labeled tags, or acoustically. Recently, MEMS based sensors have been incorporated in the biotechnical and biomedical fields. Application of acoustic biosensors include cell detection, glucose biosensing, antibody-antigen recognition, and protein adsorption detection.
Piezoelectric quartz crystal microbalances (QCMs) have been used since the late 1950s to detect gas and liquid phase analytes. Application of QCM technology to biological analytes is more recent. QCMs have been used to track the non-specific adsorption of proteins to unmodified and modified quartz crystal surface electrodes. Immobilization of antibodies to the crystal surface confers analyte specificity.
What is needed, is an apparatus that allows an architecture for constructing a solid-state biosensor for label-free detection of VEGF hybridization.